Therapeutic applications of smad7

ABSTRACT

The invention provides methods and compositions for the treatment of inflammatory and/or tissue damage conditions. In particular, the use of Smad7 compositions delivered locally or systemically to a site of inflammation and/or tissue damage is described. Other specific embodiments concern treatment or prevention of side effects caused by radiation and/or chemotherapy, including but not limited to mucositis.

The present application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 61/385,445, filed Sep. 22, 2010, the entirecontents of which are hereby incorporated by reference.

FEDERALLY FUNDED RESEARCH

Embodiments of the present invention were supported in part by grantnumber GM70966 awarded by the National Institutes of Health. The U.S.government has certain rights to the referenced invention.

FIELD

The present invention relates general to the fields of oncology andcancer therapies, inflammatory diseases and chronic wound healing. Morespecifically, the invention relates to methods and compositions for thetreatment of inflammatory diseases, chronic wound healing/ulceration,and side effects resulting from chemotherapy and radiation therapy, aswell as overexposure to radiation in military/industrial/safety/rescuepersonnel. In certain embodiments, side effects can include oralmucositis, gut mucositis and bone marrow failure. In particularembodiments, the use of Smad7 (mothers against decapentaplegic-7)protein compositions to prevent or treat the indications described aboveis provided.

BACKGROUND

Inflammation is a protective attempt by the organism to remove theinjurious stimuli and to initiate the healing process. Withoutinflammation, wounds and infections would never heal. Similarly,progressive destruction of the tissue would compromise the survival ofthe organism. However, chronic inflammation can also lead to a host ofdiseases, such as hay fever, atherosclerosis, rheumatoid arthritis,psoriasis and even cancer (e.g., gall bladder carcinoma), and acuteinflammation may cause injury through overresponse to an acute stimulus.It is for that reason that inflammation is normally closely regulated bythe body.

As indicated above, inflammation can be classified as either acute orchronic. Acute inflammation is the initial response of the body toharmful stimuli and is achieved by the increased movement of plasma andleukocytes (especially granulocytes) from the blood into the injuredtissues. A cascade of biochemical events propagates and matures theinflammatory response, involving the local vascular system, the immunesystem, and various cells within the injured tissue. Prolongedinflammation, known as chronic inflammation, leads to a progressiveshift in the type of cells present at the site of inflammation and ischaracterized by simultaneous destruction and healing of the tissue fromthe inflammatory process.

Acute inflammation begins within seconds to minutes following the injuryof tissues. The damage may be purely physical, or it may involve theactivation of an immune response. Three main processes occur: (a)increased blood flow due to dilation of blood vessels (arterioles)supplying the region; (b) increased permeability of the capillaries,allowing fluid and blood proteins to move into the interstitial spaces;and (c) migration of neutrophils (and perhaps a few macrophages) out ofthe venules and into interstitial spaces

One particular type of inflammatory injury is that stemming from the useof therapeutic agents, many of which are intended to produceinflammation in a diseased tissue or organ, but which unfortunately arerarely able to specifically target those regions. For example, over 80%of oral cancer patients are treated with radiation therapy and at least75% of these individuals will develop oral mucositis. Oral mucositisalso occurs in patients undergoing a hematopoietic stem cell transplantand in other cancer patients requiring radiotherapy and/or chemotherapy,and is often the most severe complication of radiotherapy. Severe oralmucositis is extremely painful and impairs oral intake. Subjectssuffering from oral mucositis often require long-term pain medicationsto alleviate the symptoms of this condition. In addition toinflammation, both chemo- and radiation-induced DNA damage and celldeath can bone marrow failure, resulting in death. These are is just twoexamples, if better regulated, could not only save substantially onhealthcare expense, but prevent considerable suffering, loss of patientfunction, death.

SUMMARY OF THE INVENTION

Thus, in accordance with the present invention, there is provided amethod for treating an inflammatory condition and/or tissue damageconditions in a subject comprising providing to the subject atherapeutically effective amount of a Smad7 (mothers againstdecapentaplegic homolog 7). The Smad7 may be provided as a protein, anexpression vector encoding a Smad7 protein, such as a viral or non-viralvector. The composition may comprise a Smad7 protein fused to a proteintransduction domain (PTD), and said Smad7 protein fusion may furthercomprises a regulatory domain that prevents Smad7 function in theabsence of an activator, and the method further comprises provision ofsaid activator. The Smad7 may be provided in a formulation on a patch,in a gelatinous composition, in a microsphere, in a microbead orcombination thereof. The patch may comprise a biodegradable patch, suchas an alginate polymer.

The Smad7 may be provided local to an affected region or systemically.The method may further comprise administering a second mucositis therapyto said subject, such as viscous 2% lidocaine, baking soda solution,saline solution, BAX solution (lidocaine, diphenhyramine, sorbitol andMylanta), beta carotene, tocopherol, laser irradiation, silver-nitrate,misoprostol, leucovorin, systemic keratinocyte growth factor,pentoxifylline, allopurinol, systemic sucralfate, chlorhexidinegluconate or cryotherapy. The composition may comprise a detectablemarker. The inflammatory condition is selected from mucositis,psoriasis, an autoimmune disease, chronic wound, trauma, chemotherapy,radiotherapy or cytokine therapy. The method may further compriseadministering said composition to said subject a second time.

In another embodiment, there is provided a method for treating orpreventing oral mucositis in a subject undergoing radiation therapyand/or chemotherapy comprising providing to the subject atherapeutically effective amount of a Smad7 (mothers againstdecapentaplegic homolog 7). The Smad7 may be provided as a protein, anexpression vector encoding a Smad7 protein, such as a viral or non-viralvector. The composition may comprise a Smad7 protein fused to a proteintransduction domain (PTD), and said Smad7 protein fusion may furthercomprises a regulatory domain that prevents Smad7 function in theabsence of an activator, and the method further comprises provision ofsaid activator. The Smad7 may be provided in a formulation on a patch,in a gelatinous composition, in a microsphere, in a microbead orcombination thereof. The patch may comprise a biodegradable patch, suchas an alginate polymer.

The Smad7 may be provided local to an affected region or systemically.The method may further comprise administering a second mucositis therapyto said subject, such as viscous 2% lidocaine, baking soda solution,saline solution, BAX solution (lidocaine, diphenhyramine, sorbitol andMylanta), beta carotene, tocopherol, laser irradiation, silver-nitrate,misoprostol, leucovorin, systemic keratinocyte growth factor,pentoxifylline, allopurinol, systemic sucralfate, chlorhexidinegluconate or cryotherapy. The subject may comprises a subject having atransplant, a subject having cancer or a subject having a conditionrequiring radiation therapy, such where the subject has cancer and thecancer is selected from the group consisting of oral cancer, coloncancer, breast cancer, head and neck cancer, pancreatic cancer and othercancers treated with upper body radiation or repeated cycles ofchemotherapy. The subject may have undergone upper-body radiation. Themethod may further comprise administering said composition to saidsubject a second time.

In yet another embodiment, there is provided a kit for treating oralmucositis in a subject comprising (a) a Smad7 agent composition; and (b)a delivery system. The agent may comprises Smad7 protein, optionallyfused to a protein transduction domain (PTD), and/or optionally fused toa regulatory domain that prevents Smad7 function in the absence of anactivator, and said kit further comprises said activator. The agent maybe a Smad7 expression vector, such as a viral or a non-viral expressionvector. The delivery system may comprise a gel, salve or patch deliverysystem. The composition may be lyophilized. The kit may further comprisea pharmaceutically acceptable buffer, solvent or diluent.

Some embodiments concern upregulation of TGF-β and/or NF-κB inhibitorymolecules in a subject having or suspected of developing oral mucositisor psoriasis. In certain embodiments, compositions and methods hereinconcern, induction of Smad7 (mothers against decapentaplegic homolog 7)genes or protein levels or introduction of a Smad7 composition to asubject to treat a subject having a condition disclosed herein. Otherembodiments concern over-expression of Smad7 (mothers againstdecapentaplegic homolog 7) genes and/or induction of Smad7 proteinlevels in a subject in need of such a treatment. Some embodimentsconcern using compositions of recombinant forms of Smad7 for expressionof Smad7 in a subject.

Other embodiments herein concern treating or preventing oral mucositisin a subject having undergone or that will undergo radiation therapy orchemotherapy. In certain embodiments, these subjects are undergoingtreatment for cancer including, but not limited to, oral cancer, headand neck cancer, and other cancers treated with repeated cycles ofchemotherapy including, but not limited to, colon cancer, breast cancer,pancreatic cancer, and other cancers.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments of the presentinvention. The embodiments may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIG. 1. Resistance to radiation-induced oral mucositis in Smad7transgenic mice. Dotted lines in H&E highlight the epithelial-stromalboundary. Smad7 mice had an intact oral epithelial layer while wild-type(wt) mice had oral ulcers. pH2AX, a marker for DNA damage (green), wasobvious in wt epithelium (K14, red) of oral mucositis, but wassignificantly reduced in Smad7 transgenic lesion. CD45, stained fortotal leukocytes (brown), showed severe inflammation in wild-type lesionbut a significant reduction in Smad7 transgenic lesion. CD45immunostaining has non-specific staining in the stratum corneum. Forimmunostaining, wild-type epithelial images were taken adjacent to theulcer and Smad7 images were taken from an area with the most leukocytesclose to the basement membrane.

FIG. 2. Smad7 transgene expression reversed psoriasis of K5.TGFβ1^(wt)mice. Upper panel: gross appearance of a 3 month-oldK5.TGFβ1^(wt)/K5.Smad7 mouse (smooth skin) and its K5.TGFβ1^(wt)littermate (inflamed skin, eyes and ears). Lower panel: whileK5.TGFβ1^(wt) skin exhibited epidermal hyperplasia and skininflammation, K5.Smad7 skin and K5.Smad7/TGFβ1^(wt) skin are similar tonormal wild-type (WT) skin.

FIG. 3. Tat-Smad7 protein transduction to keratinocytes. Left and middlepanels: Tat-Smad7 staining (green) in transduced cells, counterstainedby a keratin K14 antibody (red). Right: pSmad2 (green) stainsvehicle-treated (control) keratinocytes but not in Tat-Smad7 transducedcells. Tat-Smad7 staining using a V5 antibody showed nuclear and/orcytoplasmic staining of transduced cells but not in control cells.

FIG. 4. Local Tat-Smad7 protein delivery to oral mucosa. Left: PBSbuffer treated buccal mucosa. Right: buccal mucosa 24 h after oralTat-Smad7 treatment. Tat-Smad7 was stained using a V5 antibody(counterstained by a K14 antibody).

FIG. 5. Abrogation of skin inflammation by Tat-Smad7 protein. Upper:K5.TGFβ1 mouse gross photo and sections of his skin biopsy prior totreatment with thickened (wrinkled) skin and rash due to severeinflammation. Lower: the same mouse 27 days after treatment showssignificant improvement with reduced pSmad2 and NFκB p50. Vehicletreated mice have no effects (not shown). Dotted lines highlight theepidermis.

FIG. 6. Accelerated body weight recovery by orally treating Tat-Smad7protein after 20Gy irradiation. Daily Tat-Smad7 treatment began on day 5after radiation. Irradiation with 16Gy gives similar results (notshown). *: p<0.05.

FIG. 7. Oral Tat-Smad7 treatment accelerated healing of oral mucositis.Black dotted lines on top panels highlight the center of damaged areasand white dotted lines highlight the epithelial-stromal boundary. Noteoral mucosa treated with vehicle control developed oral mucositis withcomplete loss of epithelial layer on day 9 (D9) after 20Gy radiationwhereas Tat-Smad7 treated mucosa retain thin layers of oral epitheliumand numerous infiltrated leukocytes. By day 14, control mucosa hasfinished re-epithelialization with thin, undifferentiated epithelium andnumerous leukocytes in the stroma, whereas Tat-Smad7 treated mucosa hasalmost completely recovered morphology and differentiation. Radiationwith 16Gy induced oral epithelial thinning on day 9 in control mice butnot in Tat-Smad7 treated oral epithelium.

FIG. 8. Tat-Smad7 abrogates damage in radiation-induced oral mucositis.All sections are from oral mucosa immediately adjacent to the center ofthe damaged areas on day 14 after 20Gy irradiation, except CD45staining, which are selected from the most damaged areas on day 9 afterirradiation. K14 antibody was used to counterstain of epithelial layersexcept in Tunel assay.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As discussed above, over 80% of oral cancer patients are treated withradiation and at least 80% of these individuals develop oral mucositis.In addition, at least 40% and up to 70% of individuals treated withstandard chemotherapy regimens or upper-body radiation develop oralmucositis. For example, ˜70% colon cancer patients develop severe oralmucositis due to repeated chemotherapy. Hence, a treatment for oralmucositis would be relevant to millions of patients in the United Statesalone. At present, there remains no truly effective therapy for oralmucositis in cancer patients. Other inflammatory disease states havesimilar needs for improved therapies.

The Smad7 therapy disclosed below will have a significant therapeuticeffect on oral mucositis as well as other chronic, acute orperiodic/intermittent inflammatory states. Because this protein has amuch stronger effect on inflammation and better promotes wound healingcompared to current treatments (e.g. Kepivance®), and because a topicaltreatment can be user-friendly for example (application of oralsolution, gels or patches by patients), these treatments represent aconsiderable advance. Indeed, since severe oral mucositis patients oftenrequire extended hospitalization to manage feeding tubes, dehydrationand administering opioids for pain control, the embodiments describedherein represent a substantial opportunity to improve the quality oflife for patients and significantly reduce the burden of medical cost.

This approach for using Smad7 as a therapeutic agent for oral mucositisis completely unique. In particular, the inventors propose that Smad7protein can be easily produced recombinantly and used directly orencapsulated into delivery vehicles such as patches, solutions and gelsthat transduce target cells and exert bioactivities. In addition, thistype of Smad7 delivery and thus should show therapeutic efficacies withlittle side effects. However, viral vector delivery, as well as in vivoinduction and activation of Smad7, also are contemplated.

In accordance with various embodiments of the present invention, theremay be employed conventional molecular biology, microbiology, andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. See, e.g., Sambrook et al.(1990).

A. SMAD7 COMPOSITIONS

Mothers against decapentaplegic homolog 7 (Smad7) was previouslyidentified as an antagonist of TGF-β signaling by several mechanismsincluding: (a) blockade of TGF-β receptor-mediated phosphorylation andnuclear translocation of signaling Smads; (b) increased degradation ofTGF-β receptors and signaling Smads through specificubiquitin-proteasome pathways and (c) inhibition of signaling Smads fortheir binding to Smad binding elements (SBEs). Smad7 also antagonizesother signaling pathways, like the NF-κB pathway.

Smad7 protein is encoded by the SMAD7 gene (SEQ ID NOS: 1 and 2). Likemany other TGF-β family members, Smad7 is involved in cell signalling.It is a TGF-β type 1 receptor antagonist. It blocks TGF-β1 and activinassociating with the receptor, blocking access to Smad2. It is aninhibitory Smad (1-SMAD) and is enhanced by SMURF2. Smad7 also enhancesmuscle differentiation.

In one embodiment, the present invention relates to Smad7 proteincompositions. In addition to the entire Smad7 molecule, the presentinvention also relates to fragments of the polypeptide that retain theanti-inflammatory activity. Fragments may be generated by geneticengineering of translation stop sites within the coding region(discussed below). Alternatively, treatment of the Smad7 molecule withproteolytic enzymes, known as proteases, can produces a variety ofN-terminal, C-terminal and internal fragments. These fragments may bepurified according to known methods, such as precipitation (e.g.,ammonium sulfate), HPLC, ion exchange chromatography, affinitychromatography (including immunoaffinity chromatography) or various sizeseparations (sedimentation, gel electrophoresis, gel filtration).

Variants of Smad7 are also contemplated—these can be substitutional,insertional or deletion variants. Deletion variants lack one or moreresidues of the native protein which are not essential for activity,including the truncation mutants described above. Substitutionalvariants typically contain the exchange of one amino acid for another atone or more sites within the protein, and may be designed to modulateone or more properties of the polypeptide, such as stability againstproteolytic cleavage, without the loss of other functions or properties.Substitutions of this kind preferably are conservative, that is, oneamino acid is replaced with one of similar shape and charge.Conservative substitutions are well known in the art and include, forexample, each amino acid can be changed or substituted with a differentamino acid. In making substitutional variants, the hydropathic index,hydrophilicity, charge and size are normally considered.

A specialized kind of variant is the fusion protein. This moleculegenerally has all or a substantial portion of the native molecule,linked at the N- or C-terminus, to all or a portion of a secondpolypeptide. For example, fusions may employ leader sequences from otherspecies to permit the recombinant expression of a protein in aheterologous host. Another useful fusion includes the addition of afunctionally active domain, such as an antibody epitope, to facilitatepurification of the fusion protein. Another type of fusion includesattaching a domain that can act as the target for an activating orinactivating ligand, thereby permitting control of the fusion protein'sfunction once delivered to a subject. Such domains include, for example,steroid ligand binding (e.g., ER, PR, GR), which can be activated bysmall molecules, e.g., 4-hydroxyl tomaxifen or RU486 that are eitheruniquely able to activate those steroid ligand binding domains and/or donot exist in nature and will therefore enable full control of the Smad7function by the presence of these small molecules.

Another specific form of a fusion protein finding particular utility inthe present invention is a fusion including a protein transductiondomain (PTD), also called a cell delivery domain or cell transductiondomain. Such domains have been described in the art and are generallycharacterized as short amphipathic or cationic peptides and peptidederivatives, often containing multiple lysine and arginine resides(Fischer, 2007). Perhaps the best known PTD is the TAT protein from HIV,as well HSV VP16. Other examples are shown in Table 1, below.

TABLE 1 PROTEIN TRANSDUCTION DOMAINS GALFLGWLGAAGSTMGAKKKRKV  8RQIKIWFQNRRMKWKK  9 RRMKWKK 10 RRWRRWWRRWWRRWRR 11 RGGRLSYSRRRFSTSTGR 12YGRKKRRQRRR 13 RKKRRQRRR 14 YARAAARQARA 15 RRRRRRRR 16 KKKKKKKK 17GWTLNSAGYLLGKINLKALAALAKXIL 18 LLILLRRRIRKQANAHSK 19 SRRHHCRSKAKRSRHH 20NRARRNRRRVR 21 RQLRIAGRRLRGRSR 22 KLIKGRTPIKFGK 23 RRIPNRRPRR 24KLALKLALKALKAALKLA 25 KLAKLAKKLAKLAK 26 GALFLGFLGAAGSTNGAWSQPKKKRKV 27KETWWETWWTEWSQPKKKRKV 28 LKKLLKKLLKKLLKKLLKKL 29 SEQ ID NO:QAATATRGRSAASRPTERPRAPARSASRPRRPVE 30 MGLGLHLLVLAAALQGAKSKRKV 31AAVALLPAVLLALLAPAAANYKKPKL 32 MANLGYWLLALFVTMWTDVGLCKKRPKP 33LGTYTQDFNKFHTFPQTAIGVGAP 34 DPKGDPKGVTVTVTVTVTGKGDPXPD 35 PPPPPPPPPPPPPP36 VRLPPPVRLPPPVRLPPP 37 PRPLPPPRPG 38 SVRRRPRPPYLPRPRPPPFFPPRLPPRIPP 39TRSSRAGLQFPVGRVHRLLRK 40 GIGKFLHSAKKFGKAFVGEIMNS 41KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK 42 ALWMTLLKKVLKAAAKAALNAVLVGANA 43GIGAVLKVLTTGLPALISWIKRKRQQ 44 INLKALAALAKKIL 45GFFALIPKIISSPLPKTLLSAVGSALGGSGGQE 46 LAKWALKQGFAKLKS 47SMAQDIISTIGDLVKWIIQTVNXFTKK 48 LLGDFFRKSKEKIGKEFKRIVQRIKQRIKDFLANLVPRTES49 PAWRKAFRWAWRMLKKAA 50 KLKLKLKLKLKLKLKLKL 51

B. NUCLEIC ACIDS, VECTORS AND RECOMBINANT EXPRESSION

The present invention also provides, in another embodiment, genesencoding Smad7. In addition to the identified SMAD7 gene (SEQ ID NO: 2),it should be clear that the present invention is not limited to thespecific nucleic acids disclosed herein. As discussed below, a “Smad7gene” may contain a variety of different bases and yet still produce acorresponding polypeptide that is functionally indistinguishable from,and in some cases structurally identical to, the human gene disclosedherein.

1. Nucleic Acids Encoding Smad7

Nucleic acids according to the present invention may represent an entireSmad7 gene, a domain of Smad7 that expresses a tumor suppressingfunction, or any other fragment of the Smad7 sequences set forth herein.The nucleic acid may be derived from genomic DNA, i.e., cloned directlyfrom the genome of a particular organism. In preferred embodiments,however, the nucleic acid would comprise complementary DNA (cDNA). Alsocontemplated is a cDNA plus a natural intron or an intron derived fromanother gene; such engineered molecules are sometime referred to as“mini-genes.” At a minimum, these and other nucleic acids of the presentinvention may be used as molecular weight standards in, for example, gelelectrophoresis.

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression or where non-coding regions such asintrons are to be targeted in an antisense strategy.

As used in this application, the term “a nucleic acid encoding a Smad7”refers to a nucleic acid molecule that has been isolated free of totalcellular nucleic acid. In certain embodiments, the invention concerns anucleic acid sequence essentially as set forth in SEQ ID NO:2. The term“as set forth in SEQ ID NO:2” means that the nucleic acid sequencesubstantially corresponds to a portion of SEQ ID NO:2. The term“functionally equivalent codon” is used herein to refer to codons thatencode the same amino acid, such as the six codons for arginine orserine, and also refers to codons that encode biologically equivalentamino acids, as discussed in the following pages.

Allowing for the degeneracy of the genetic code, sequences that have atleast about 50%, usually at least about 60%, more usually about 70%,most usually about 80%, preferably at least about 90% and mostpreferably about 95% of nucleotides that are identical to thenucleotides of SEQ ID NO:2. Sequences that are essentially the same asthose set forth in SEQ ID NO:2 also may be functionally defined assequences that are capable of hybridizing to a nucleic acid segmentcontaining the complement of SEQ ID NO:2 under standard conditions.

The DNA segments of the present invention include those encodingbiologically functional equivalent Smad7 proteins and peptides, asdescribed above. Such sequences may arise as a consequence of codonredundancy and amino acid functional equivalency that are known to occurnaturally within nucleic acid sequences and the proteins thus encoded.Alternatively, functionally equivalent proteins or peptides may becreated via the application of recombinant DNA technology, in whichchanges in the protein structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by man may be introduced through the application ofsite-directed mutagenesis techniques or may be introduced randomly andscreened later for the desired function, as described below.

2. Vectors for Cloning, Gene Transfer and Expression

Within certain embodiments, expression vectors are employed to expressthe Smad7 polypeptide product, which can then be purified for varioususes. In other embodiments, the expression vectors are used in genetherapy. Expression requires that appropriate signals be provided in thevectors, and which include various regulatory elements, such asenhancers/promoters from both viral and mammalian sources that driveexpression of the genes of interest in host cells. Elements designed tooptimize messenger RNA stability and translatability in host cells alsoare defined. The conditions for the use of a number of dominant drugselection markers for establishing permanent, stable cell clonesexpressing the products are also provided, as is an element that linksexpression of the drug selection markers to expression of thepolypeptide.

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. In certain embodiments,expression includes both transcription of a gene and translation of mRNAinto a gene product. In other embodiments, expression only includestranscription of the nucleic acid encoding a gene of interest.

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques, which are described in Sambrook et al. (1989) and Ausubel etal. (1994), both incorporated herein by reference.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host organism, including promoters andenhancers. In addition to control sequences that govern transcriptionand translation, vectors and expression vectors may contain nucleic acidsequences that serve other functions, such as transcription terminationsignals and poly-adenylation sites.

The capacity of certain viral vectors to efficiently infect or entercells, to integrate into a host cell genome and stably express viralgenes, have led to the development and application of a number ofdifferent viral vector systems (Robbins et al., 1998). Viral systems arecurrently being developed for use as vectors for ex vivo and in vivogene transfer. For example, adenovirus, herpes-simplex virus,lentiviruses, retrovirus and adeno-associated virus vectors are beingevaluated currently for treatment of diseases such as cancer, cysticfibrosis, Gaucher disease, renal disease and arthritis (Robbins andGhivizzani, 1998; Imai et al., 1998; U.S. Pat. No. 5,670,488). Thevarious viral vectors present specific advantages and disadvantages,depending on the particular gene-therapeutic application.

Suitable non-viral methods for nucleic acid delivery for transformationof an organelle, a cell, a tissue or an organism for use with thecurrent invention are believed to include virtually any method by whicha nucleic acid (e.g., DNA) can be introduced into an organelle, a cell,a tissue or an organism, as described herein or as would be known to oneof ordinary skill in the art. Such methods include, but are not limitedto, direct delivery of DNA such as by injection (U.S. Pat. Nos.5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932,5,656,610, 5,589,466 and 5,580,859, each incorporated herein byreference), including microinjection (Harland and Weintraub, 1985; U.S.Pat. No. 5,789,215, incorporated herein by reference); byelectroporation (U.S. Pat. No. 5,384,253, incorporated herein byreference); by calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE-dextranfollowed by polyethylene glycol (Gopal, 1985); by direct sonic loading(Fechheimer et al., 1987); by liposome mediated transfection (Nicolauand Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al.,1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectilebombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat.Nos. 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and5,538,880, and each incorporated herein by reference); by agitation withsilicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523and 5,464,765, each incorporated herein by reference); or byPEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S.Pat. Nos. 4,684,611 and 4,952,500, each incorporated herein byreference); by desiccation/inhibition-mediated DNA uptake (Potrykus etal., 1985). Through the application of techniques such as these,organelle(s), cell(s), tissue(s) or organism(s) may be stably ortransiently transformed.

5. Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with the present invention to producenucleic acid sequences, or their cognate polypeptides, proteins andpeptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986 and 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MAXBAC®2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROMCLONTECH®.

Other examples of expression systems include STRATAGENE®'S COMPLETECONTROL™ Inducible Mammalian Expression System, which involves asynthetic ecdysone-inducible receptor, or its pET Expression System, anE. coli expression system. Another example of an inducible expressionsystem is available from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

Primary mammalian cell cultures may be prepared in various ways. Inorder for the cells to be kept viable while in vitro and in contact withthe expression construct, it is necessary to ensure that the cellsmaintain contact with the correct ratio of oxygen and carbon dioxide andnutrients but are protected from microbial contamination. Cell culturetechniques are well documented.

One embodiment of the foregoing involves the use of gene transfer toimmortalize cells for the production of proteins. The gene for theprotein of interest may be transferred as described above intoappropriate host cells followed by culture of cells under theappropriate conditions. The gene for virtually any polypeptide may beemployed in this manner. The generation of recombinant expressionvectors, and the elements included therein, are discussed above.Alternatively, the protein to be produced may be an endogenous proteinnormally synthesized by the cell in question.

Examples of useful mammalian host cell lines are Vero and HeLa cells andcell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2,NIH3T3, RIN and MDCK cells. In addition, a host cell strain may bechosen that modulates the expression of the inserted sequences, ormodifies and process the gene product in the manner desired. Suchmodifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to insure the correctmodification and processing of the foreign protein expressed.

A number of selection systems may be used including, but not limited to,HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase andadenine phosphoribosyltransferase genes, in tk−, hgprt− or aprt− cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to; gpt, that confersresistance to mycophenolic acid; neo, that confers resistance to theaminoglycoside G418; and hygro, that confers resistance to hygromycin.

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organisms that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny.

Host cells may be derived from prokaryotes or eukaryotes, depending uponwhether the desired result is replication of the vector or expression ofpart or all of the vector-encoded nucleic acid sequences. Numerous celllines and cultures are available for use as a host cell, and they can beobtained through the American Type Culture Collection (ATCC), which isan organization that serves as an archive for living cultures andgenetic materials (atcc.org). An appropriate host can be determined byone of skill in the art based on the vector backbone and the desiredresult. A plasmid or cosmid, for example, can be introduced into aprokaryote host cell for replication of many vectors. Bacterial cellsused as host cells for vector replication and/or expression includeDH5α, JM109, and KC8, as well as a number of commercially availablebacterial hosts such as SURE® Competent Cells and SOLOPACK™ Gold Cells(STRATAGENE®, La Jolla). Alternatively, bacterial cells such as E. coliLE392 could be used as host cells for phage viruses.

Examples of eukaryotic host cells for replication and/or expression of avector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Manyhost cells from various cell types and organisms are available and wouldbe known to one of skill in the art. Similarly, a viral vector may beused in conjunction with either a eukaryotic or prokaryotic host cell,particularly one that is permissive for replication or expression of thevector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

C. INFLAMMATORY DISEASE STATES

1. Chronic Wounds

A chronic wound is a wound that does not heal in an orderly set ofstages and in a predictable amount of time the way most wounds do;wounds that do not heal within three months are often consideredchronic. Chronic wounds seem to be detained in one or more of the phasesof wound healing. For example, chronic wounds often remain in theinflammatory stage for too long. In acute wounds, there is a precisebalance between production and degradation of molecules such ascollagen; in chronic wounds this balance is lost and degradation playstoo large a role.

Chronic wounds may never heal or may take years to do so. These woundscause patients severe emotional and physical stress as well as creatinga significant financial burden on patients and the whole healthcaresystem. Acute and chronic wounds are at opposite ends of a spectrum ofwound healing types that progress toward being healed at differentrates. The vast majority of chronic wounds can be classified into threecategories: venous ulcers, diabetic, and pressure ulcers. A small numberof wounds that do not fall into these categories may be due to causessuch as radiation poisoning or ischemia.

Venous and Arterial Ulcers.

Venous ulcers, which usually occur in the legs, account for about 70% to90% of chronic wounds and mostly affect the elderly. They are thought tobe due to venous hypertension caused by improper function of valves thatexist in the veins to prevent blood from flowing backward. Ischemiaresults from the dysfunction and, combined with reperfusion injury,causes the tissue damage that leads to the wounds.

Diabetic Ulcers.

Another major cause of chronic wounds, diabetes, is increasing inprevalence. Diabetics have a 15% higher risk for amputation than thegeneral population due to chronic ulcers. Diabetes causes neuropathy,which inhibits nociception and the perception of pain. Thus patients maynot initially notice small wounds to legs and feet, and may thereforefail to prevent infection or repeated injury. Further, diabetes causesimmune compromise and damage to small blood vessels, preventing adequateoxygenation of tissue, which can cause chronic wounds. Pressure alsoplays a role in the formation of diabetic ulcers.

Pressure Ulcers.

Another leading type of chronic wounds is pressure ulcers, which usuallyoccur in people with conditions such as paralysis that inhibit movementof body parts that are commonly subjected to pressure such as the heels,shoulder blades, and sacrum. Pressure ulcers are caused by ischemia thatoccurs when pressure on the tissue is greater than the pressure incapillaries, and thus restricts blood flow into the area. Muscle tissue,which needs more oxygen and nutrients than skin does, shows the worsteffects from prolonged pressure. As in other chronic ulcers, reperfusioninjury damages tissue.

Chronic wounds may affect only the epidermis and dermis, or they mayaffect tissues all the way to the fascia. They may be formed originallyby the same things that cause acute ones, such as surgery or accidentaltrauma, or they may form as the result of systemic infection, vascular,immune, or nerve insufficiency, or comorbidities such as neoplasias ormetabolic disorders. The reason a wound becomes chronic is that thebody's ability to deal with the damage is overwhelmed by factors such asrepeated trauma, continued pressure, ischemia, or illness.

Though much progress has been accomplished in the study of chronicwounds lately, advances in the study of their healing have lagged behindexpectations. This is partly because animal studies are difficultbecause animals do not get chronic wounds, since they usually have looseskin that quickly contracts, and they normally do not get old enough orhave contributing diseases such as neuropathy or chronic debilitatingillnesses. Nonetheless, current researchers now understand some of themajor factors that lead to chronic wounds, among which are ischemia,reperfusion injury, and bacterial colonization.

Ischemia.

Ischemia is an important factor in the formation and persistence ofwounds, especially when it occurs repetitively (as it usually does) orwhen combined with a patient's old age. Ischemia causes tissue to becomeinflamed and cells to release factors that attract neutrophils such asinterleukins, chemokines, leukotrienes, and complement factors.

While they fight pathogens, neutrophils also release inflammatorycytokines and enzymes that damage cells. One of their important jobs isto produce Reactive Oxygen Species (ROS) to kill bacteria, for whichthey use an enzyme called myeloperoxidase. The enzymes and ROS producedby neutrophils and other leukocytes damage cells and prevent cellproliferation and wound closure by damaging DNA, lipids, proteins, theECM, and cytokines that speed healing. Neutrophils remain in chronicwounds for longer than they do in acute wounds, and contribute to thefact that chronic wounds have higher levels of inflammatory cytokinesand ROS. Since wound fluid from chronic wounds has an excess ofproteases and ROS, the fluid itself can inhibit healing by inhibitingcell growth and breaking down growth factors and proteins in the ECM.

Bacterial Colonization.

Since more oxygen in the wound environment allows white blood cells toproduce ROS to kill bacteria, patients with inadequate tissueoxygenation, for example those who suffered hypothermia during surgery,are at higher risk for infection. The host's immune response to thepresence of bacteria prolongs inflammation, delays healing, and damagestissue. Infection can lead not only to chronic wounds but also togangrene, loss of the infected limb, and death of the patient.

Like ischemia, bacterial colonization and infection damage tissue bycausing a greater number of neutrophils to enter the wound site. Inpatients with chronic wounds, bacteria with resistances to antibioticsmay have time to develop. In addition, patients that carry drugresistant bacterial strains such as methicillin-resistant Staphylococcusaureus (MRSA) have more chronic wounds.

Growth Factors and Proteolytic Enzymes.

Chronic wounds also differ in makeup from acute wounds in that theirlevels of proteolytic enzymes such as elastase. and matrixmetalloproteinases (MMPs) are higher, while their concentrations ofgrowth factors such as Platelet-derived growth factor and KeratinocyteGrowth Factor are lower.

Since growth factors (GFs) are imperative in timely wound healing,inadequate GF levels may be an important factor in chronic woundformation. In chronic wounds, the formation and release of growthfactors may be prevented, the factors may be sequestered and unable toperform their metabolic roles, or degraded in excess by cellular orbacterial proteases.

Chronic wounds such as diabetic and venous ulcers are also caused by afailure of fibroblasts to produce adequate ECM proteins and bykeratinocytes to epithelialize the wound. Fibroblast gene expression isdifferent in chronic wounds than in acute wounds.

Though all wounds require a certain level of elastase and proteases forproper healing, too high a concentration is damaging. Leukocytes in thewound area release elastase, which increases inflammation, destroystissue, proteoglycans, and collagen, and damages growth factors,fibronectin, and factors that inhibit proteases. The activity ofelastase is increased by human serum albumin, which is the most abundantprotein found in chronic wounds. However, chronic wounds with inadequatealbumin are especially unlikely to heal, so regulating the wound'slevels of that protein may in the future prove helpful in healingchronic wounds.

Excess matrix metalloproteinases, which are released by leukocytes, mayalso cause wounds to become chronic. MMPs break down ECM molecules,growth factors, and protease inhibitors, and thus increase degradationwhile reducing construction, throwing the delicate compromise betweenproduction and degradation out of balance.

2. Acute Wounds/Trauma

Physical trauma is a serious and body-altering physical injury, such asthe removal of a limb. Blunt force trauma, a type of physical traumacaused by impact or other force applied from or with a blunt object,whereas penetrating trauma is a type of physical trauma in which theskin or tissues are pierced by an object. Trauma can also be describedas both unplanned, such as an accident, or planned, in the case ofsurgery. Both can be characterized by mild to severe tissue damage,blood loss and/or shock, and both may lead to subsequent infection,including sepsis. The present invention provides to treatment of trauma,including both pre-treatment (in the case of a medical procedure) andtreatment after trauma injury as occurred.

Surgery.

Surgery uses operative manual and instrumental techniques on a patientto investigate and/or treat a pathological condition such as disease orinjury, to help improve bodily function or appearance, or sometimes forsome other reason. The present invention can address trauma resultingfrom surgeries, as defined further below.

As a general rule, a procedure is considered surgical when it involvescutting of a patient's tissues or closure of a previously sustainedwound. Other procedures that do not necessarily fall under this rubric,such as angioplasty or endoscopy, may be considered surgery if theyinvolve common surgical procedure or settings, such as use of a sterileenvironment, anesthesia, antiseptic conditions, typical surgicalinstruments, and suturing or stapling. All forms of surgery areconsidered invasive procedures; so-called noninvasive surgery usuallyrefers to an excision that does not penetrate the structure beingaddressed (e.g., laser ablation of the cornea) or to a radiosurgicalprocedure (e.g., irradiation of a tumor). Surgery can last from minutesto hours.

Surgical procedures are commonly categorized by urgency, type ofprocedure, body system involved, degree of invasiveness, and specialinstrumentation. Elective surgery is done to correct anon-life-threatening condition, and is carried out at the patient'srequest, subject to the surgeon's and the surgical facility'savailability. Emergency surgery is surgery which must be done quickly tosave life, limb, or functional capacity. Exploratory surgery isperformed to aid or confirm a diagnosis. Therapeutic surgery treats apreviously diagnosed condition.

Amputation involves cutting off a body part, usually a limb or digit.Replantation involves reattaching a severed body part. Reconstructivesurgery involves reconstruction of an injured, mutilated, or deformedpart of the body. Cosmetic surgery is done to improve the appearance ofan otherwise normal structure. Excision is the cutting out of an organ,tissue, or other body part from the patient. Transplant surgery is thereplacement of an organ or body part by insertion of another fromdifferent human (or animal) into the patient. Removing an organ or bodypart from a live human or animal for use in transplant is also a type ofsurgery.

When surgery is performed on one organ system or structure, it may beclassed by the organ, organ system or tissue involved. Examples includecardiac surgery (performed on the heart), gastrointestinal surgery(performed within the digestive tract and its accessory organs), andorthopedic surgery (performed on bones and/or muscles).

Minimally invasive surgery involves smaller outer incision(s) to insertminiaturized instruments within a body cavity or structure, as inlaparoscopic surgery or angioplasty. By contrast, an open surgicalprocedure requires a large incision to access the area of interest.Laser surgery involves use of a laser for cutting tissue instead of ascalpel or similar surgical instruments. Microsurgery involves the useof an operating microscope for the surgeon to see small structures.Robotic surgery makes use of a surgical robot, such as Da Vinci or Zeussurgical systems, to control the instrumentation under the direction ofthe surgeon.

3. Autoimmune/Inflammatory Disease

The present invention contemplates the treatment of a variety ofautoimmune and/or inflammatory disease states such asspondyloarthropathy, ankylosing spondylitis, psoriatic arthritis,reactive arthritis, enteropathic arthritis, ulcerative colitis, Crohn'sdisease, irritable bowel disease, inflammatory bowel disease, rheumatoidarthritis, juvenile rheumatoid arthritis, familial Mediterranean fever,amyotrophic lateral sclerosis, Sjogren's syndrome, early arthritis,viral arthritis, multiple sclerosis, or psoriasis. The diagnosis andtreatment of these diseases are well documented in the literature.

4. Chemotherapy, Radiotherapy and Cytokine Therapy Toxicity

Various forms of cancer therapy, including chemotherapy, radiation, andcytokines, are associated with toxicity, sometimes severe, in the cancerpatient. To the extent that the toxicity is caused at least in part bythe extracellular actions of histones, the present invention seeks toreduce this toxicity using the pharmaceutical compositions of thepresent invention, thereby reducing or alleviating discomfort on thepart of the patient, as well as permitting higher doses of the therapy.

Oral cancer, the 6^(th) most common cancer worldwide, is a subtype ofhead and neck cancer, is any cancerous tissue growth located in the oralcavity. It may arise as a primary lesion originating in any of the oraltissues, by metastasis from a distant site of origin, or by extensionfrom a neighboring anatomic structure, such as the nasal cavity or theoral cancers may originate in any of the tissues of the mouth, and maybe of varied histologic types: teratoma, adenocarcinoma derived from amajor or minor salivary gland, lymphoma from tonsillar or other lymphoidtissue, or melanoma from the pigment-producing cells of the oral mucosa.There are several types of oral cancers, but around 90% are squamouscell carcinomas, originating in the tissues that line the mouth andlips. Oral or mouth cancer most commonly involves the tongue. It mayalso occur on the floor of the mouth, cheek lining, gingiva (gums),lips, or palate (roof of the mouth). Most oral cancers look very similarunder the microscope and are called squamous cell carcinoma. These aremalignant and tend to spread rapidly.

Over 80% of oral cancer patients are treated with radiation therapy andat least 75% of these individuals will develop oral mucositis. Oralmucositis is a chronic oral ulceration. This disease frequently occursin radiation-treated patients of all cancer types, patientsradiation-treated for organ transplants (to eliminate rejection of thetransplants), and patients undergoing routine chemotherapy. Severe oralmucositis is extremely painful and impairs food/liquid intake, hence isoften the most severe complication of cancer therapy. Oral mucositis isa major factor in determining the maximum dose possible of radiation andchemotherapy to the head and neck region; it can significantlycomplicate cancer treatment, extend hospitalization, decrease quality oflife and increase costs. Currently, there is no established therapy toeffectively treat severe oral mucositis. To date, Kepivance®, arecombinant protein of human keratinocyte growth factor (KGF), is theonly FDA approved drug through i.v. injections for severe oral mucositisin bone-marrow transplant patients, and its use in cancer patientsremains to be determined. Hence, this drug is available for only 4% ofthe at-risk population. It also suffers from the need for medicalservice providers due to the i.v. route. Other potential therapiesinclude topical rinses, such as viscous 2% lidocaine rinses, or bakingsoda and saline solutions, or a cocktail solution, for instance BAX(lidocaine, diphenhyramine, sorbitol and Mylanta). Other investigativeor mucoprotective adjuvant therapies include but are not limited to,beta carotene, tocopherol, laser irradiation, prophylactic brushing theoral mucosa with silver-nitrate, misoprostol, leucovorin, systemic KGF,pentoxifylline, allopurinol mouthwash, systemic sucralfate,chlorhexidine gluconate, and cryotherapy.

Chemotherapy and radiation induced gut mucositits is an inflammatorycondition that arises as a result of the acute death of rapidly dividingintestinal epithelial cells. Most chemotherapeutic drugs used fortreatment of solid tumors, alone, in a combination of drugs, or withradiation, will result in the death of a large number of intestinalepithelial cells. The clinical manifestations of the ensuing mucositisinclude digestive symptoms such as nausea and vomiting, seriousdiarrhea, acute weight loss and wasting. This is fast becoming one ofthe limiting factors for administering chemotherapy for many cancerpatients. The ability of Tat-Smad7 to protect intestinal epithelialcells from either chemotherapeutic agents, radiation, or a combinationsof those, will significantly decrease the undesirable side effects ofcancer therapies, and enable more aggressive ways to treat the diseasewith existing tools.

Bone marrow failure syndromes are a set of conditions that develop whenthe hematopoietic stem cell compartment is compromised and fails to giverise to normal cell types. Bone marrow failure occurs as a result ofinherited genetic abnormalities, exposure to a noxious substance, suchas toxins, chemicals or viruses. Although the nature and identity ofenvironmental factors that can lead to the development of acquired bonemarrow failure is still not completely understood, a few factors havebeen linked to the development of acquired bone marrow failure amongmilitary personnel including exposure to mustard gas, ionizingradiation, and infectious agents such as visceral leishmaniasis orAfrican trypanosomiasis. The best approach for management of bone marrowfailure syndromes is still the transplantation of HSCs, unless asufficient number of the remaining resident BM HSCs can be spared fromthese stresses and encouraged to repopulate the hematopoieticcompartment. The modulation of Smad 7, as described here, should enablefor the deliberate protection of the remaining resident HSCs in patientsthat exhibit clinical signs consistent with bone marrow failure.

5. Combination Anti-Inflammatory Therapies

It is common in many fields of medicine to treat a disease with multipletherapeutic modalities, often called “combination therapies.”Inflammatory disease are no exception. To treat inflammatory disordersusing the methods and compositions of the present invention, one wouldgenerally contact a target cell, organ or subject with a Smad7 protein,expression construct or activator and at least one other therapy. Thesetherapies would be provided in a combined amount effective to achieve areduction in one or more disease parameter. This process may involvecontacting the cells/subjects with the both agents/therapies at the sametime, e.g., using a single composition or pharmacological formulationthat includes both agents, or by contacting the cell/subject with twodistinct compositions or formulations, at the same time, wherein onecomposition includes the Smad7 agent and the other includes the otheragent.

Alternatively, the Smad7 agent may precede or follow the other treatmentby intervals ranging from minutes to weeks. One would generally ensurethat a significant period of time did not expire between the time ofeach delivery, such that the therapies would still be able to exert anadvantageously combined effect on the cell/subject. In such instances,it is contemplated that one would contact the cell with both modalitieswithin about 12-24 hours of each other, within about 6-12 hours of eachother, or with a delay time of only about 12 hours. In some situations,it may be desirable to extend the time period for treatmentsignificantly; however, where several days (2, 3, 4, 5, 6 or 7) toseveral weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

It also is conceivable that more than one administration of either theSmad7 agent or the other therapy will be desired. Various combinationsmay be employed, where the Smad7 agent is “A,” and the other therapy is“B,” as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/B

Other combinations are contemplated. Other agents suitable for use in acombined therapy against an inflammatory disorder include steroids,glucocorticoids, non-steriodal anti-inflammatory drugs (NSAIDS;including COX-1 and COX-2 inhibitors), aspirin, ibuprofen, and naproxen.Analgesics are commonly associated with anti-inflammatory drugs butwhich have no anti-inflammatory effects. An example is paracetamol,called acetaminophen in the U.S. and sold under the brand name ofTylenol. As opposed to NSAIDS, which reduce pain and inflammation byinhibiting COX enzymes, paracetamol has recently been shown to block thereuptake of endocannabinoids, which only reduces pain, likely explainingwhy it has minimal effect on inflammation. A particular agent forcombination use is an anti-TGF-β antibody.

The skilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652, 1990. Somevariation in dosage will necessarily occur depending on the condition ofthe subject being treated. The person responsible for administrationwill, in any event, determine the appropriate dose for the individualsubject. Moreover, for human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biologics standards.

It also should be pointed out that any of the foregoing therapies mayprove useful by themselves in treating inflammation.

6. Combinations with Cancer Therapy

As discussed above, the invention has particular relevance to thetreatment of DNA damage and/or inflammation resulting from certainanti-cancer therapies. Thus, in particular, the invention may be appliedas a combination to with cancer therapies. While cancer therapiesaddress the cancer, they unfortunately cause serious side effects. Assuch, the Smad7 agents of the present invention can be usedadvantageously in combination with such cancer therapies. This processmay involve contacting the cells, organ or patient with theagents/therapies at the same time, including by contacting the cells,organ or patient with a single composition or pharmacologicalformulation that includes both agents, or with two distinct compositionsor formulations at the same time, wherein one composition includes theSmad7 agent and the other includes the other agent. Alternatively,analogous to the chart set forth above, the compositions can bedelivered at different times, including repeated doses of one or bothagents.

Agents or factors suitable for use in a combined therapy include anychemical compound or treatment method that induces DNA damage whenapplied to a cell. Such agents and factors include radiation and wavesthat induce DNA damage such as, irradiation, microwaves, electronicemissions, and the like. A variety of chemical compounds, also describedas “chemotherapeutic” or “genotoxic agents,” are intended to be of usein the combined treatment methods disclosed herein. In treating canceraccording to the invention, one would contact the tumor cells with anagent in addition to the expression construct. This may be achieved byirradiating the localized tumor site; alternatively, the tumor cells maybe contacted with the agent by administering to the subject atherapeutically effective amount of a pharmaceutical composition.

Various classes of chemotherapeutic agents are comtemplated for use within combination with peptides of the present invention. For example,selective estrogen receptor antagonists (“SERMs”), such as Tamoxifen,4-hydroxy Tamoxifen (Afimoxfene), Falsodex, Raloxifene, Bazedoxifene,Clomifene, Femarelle, Lasofoxifene, Ormeloxifene, and Toremifene.

Chemotherapeutic agents contemplated to be of use, include, e.g.,camptothecin, actinomycin-D, mitomycin C. The invention also encompassesthe use of a combination of one or more DNA damaging agents, whetherradiation-based or actual compounds, such as the use of X-rays withcisplatin or the use of cisplatin with etoposide. The agent may beprepared and used as a combined therapeutic composition, or kit, bycombining it with a MUC1 peptide, as described above.

Heat shock protein 90 is a regulatory protein found in many eukaryoticcells. HSP90 inhibitors have been shown to be useful in the treatment ofcancer. Such inhibitors include Geldanamycin,17-(Allylamino)-17-demethoxygeldanamycin, PU-H71 and Rifabutin.

Agents that directly cross-link DNA or form adducts are also envisaged.Agents such as cisplatin, and other DNA alkylating agents may be used.Cisplatin has been widely used to treat cancer, with efficacious dosesused in clinical applications of 20 mg/m² for 5 days every three weeksfor a total of three courses. Cisplatin is not absorbed orally and musttherefore be delivered via injection intravenously, subcutaneously,intratumorally or intraperitoneally.

Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for doxorubicin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally. Microtubuleinhibitors, such as taxanes, also are contemplated. These molecules arediterpenes produced by the plants of the genus Taxus, and includepaclitaxel and docetaxel.

Epidermal growth factor receptor inhibitors, such as Iressa, mTOR, themammalian target of rapamycin, also known as FK506-binding protein12-rapamycin associated protein 1 (FRAP1) is a serine/threonine proteinkinase that regulates cell growth, cell proliferation, cell motility,cell survival, protein synthesis, and transcription. Rapamycin andanalogs thereof (“rapalogs”) are therefore contemplated for use incombination cancer therapy in accordance with the present invention.

Another possible combination therapy with the peptides claimed herein isTNF-α (tumor necrosis factor-alpha), a cytokine involved in systemicinflammation and a member of a group of cytokines that stimulate theacute phase reaction. The primary role of TNF is in the regulation ofimmune cells. TNF is also able to induce apoptotic cell death, to induceinflammation, and to inhibit tumorigenesis and viral replication.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage. As such a number ofnucleic acid precursors have been developed. Particularly useful areagents that have undergone extensive testing and are readily available.As such, agents such as 5-fluorouracil (5-FU), are preferentially usedby neoplastic tissue, making this agent particularly useful fortargeting to neoplastic cells. Although quite toxic, 5-FU, is applicablein a wide range of carriers, including topical, however intravenousadministration with doses ranging from 3 to 15 mg/kg/day being commonlyused.

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, x-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageDNA, on the precursors of DNA, the replication and repair of DNA, andthe assembly and maintenance of chromosomes. Dosage ranges for x-raysrange from daily doses of 50 to 200 roentgens for prolonged periods oftime (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosageranges for radioisotopes vary widely, and depend on the half-life of theisotope, the strength and type of radiation emitted, and the uptake bythe neoplastic cells.

The skilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

In addition to combining Smad7 therapies with chemo- and radiotherapies,it also is contemplated that combination with immunotherapy, hormonetherapy, toxin therapy and surgery. In particular, one may employtargeted therapies such as Avastin, Erbitux, Gleevec, Herceptin andRituxan.

In other embodiments, to assess the roles and mechanisms of Smad7 withinthe context of oral mucositis, “gene-switch” transgenic mouse modelswere developed to allow control of the level and duration of Smad7transgene expression specifically in oral epithelia. In accordance withthese embodiments, these models may be used to test other genes ordownstream molecules for their effects on oral epithelia and oralmucosa. Thus, these models can be used for, but are not limited to,further analysis of oral wound healing biology and testing therapeuticapproaches to oral wound healing. Molecular Smad7 targets identified inthese studies can provide additional therapeutic targets for subjectssuffering from oral mucositis. Models and resources developed herein canprovide unique tools for analytical studies to identify biomarkers andtherapeutic targets related to Smad7 overexpression and control, forexample, downstream molecules turned on or bound by Smad7 can beidentified as additional therapeutic targets for example, to treat oralmucositis, psoriasis and other conditions aggravated by TGF-β activitiesand NFκB activities.

D. FORMULATIONS AND ROUTES OF DELIVERY

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions—proteins, expression vectors, virusstocks, proteins and drugs—in a form appropriate for the intendedapplication. Generally, this will entail preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal or intravenous injection. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions,described supra. Of particular interest is direct intratumoraladministration, perfusion of a tumor, or admininstration local orregional to a tumor, for example, in the local or regional vasculatureor lymphatic system, or in a resected tumor bed.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

The formulations are easily administered in a variety of dosage forms.Some variation in dosage will necessarily occur depending on thecondition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Moreover, for human administration, preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by FDA Office of Biologics standards.

For oral administration the polypeptides of the present invention may beincorporated with excipients and used in the form of non-ingestiblemouthwashes and dentifrices. A mouthwash may be prepared incorporatingthe active ingredient in the required amount in an appropriate solvent,such as a sodium borate solution (Dobell's Solution). Alternatively, theactive ingredient may be incorporated into an antiseptic wash containingsodium borate, glycerin and potassium bicarbonate. The active ingredientmay also be dispersed in dentifrices, including: gels, pastes, powdersand slurries. The active ingredient may be added in a therapeuticallyeffective amount to a paste dentifrice that may include water, binders,abrasives, flavoring agents, foaming agents, and humectants.

In accordance with these embodiments, oral delivery materials can alsoinclude creams, salves, ointments, patches, liposomes, nanoparticles,microparticles, timed-release formulations and other materials known inthe art for delivery to the oral cavity and/or to the skin of a subjectfor treatment and/or prevention of a condition disclosed herein. Certainembodiments concern using a biodegradable oral patch delivery system orgelatinous material. These compositions can be a liquid formulation or apharmaceutically acceptable delivery system treated with a formulationof these compositions, and may also include activator/inducers.

In certain embodiments, a patch contemplated herein may be a slowlydissolving or a time-released patch. In accordance with theseembodiments, a slowly dissolving patch can be an alginate patch. Incertain examples, a patch may contain a detectible indicator dye oragent such as a fluorescent agent. In other embodiments, a tag (e.g.detectible tag such as a biotin or fluorescently tagged agent) can beassociated with a treatment molecule in order to detect the moleculeafter delivery to the subject. In certain embodiments, one or more oraldelivery patches or other treatment contemplated herein may beadministered to a subject 3 times daily, 2 times daily, once a day,every other day, weekly, and the like, depending on need of the subjectassessed by a health professional. Patches contemplated herein may beoral-biodegradable patches or patches for exterior use that may or maynot degrade. Patches contemplated herein may be 1 mm, 2 mm, 3 mm, 4 mmto 5 mm in size or more depending on need. In addition, skin patches arecontemplated herein for use in a subject suffering from psoriasis. Intreating psoriasis and chronic wounds, Smad7 can be delivered topicallyusing vehicles such as glycerol, carboxymethycellulose. It can also usetransdermal system (e.g., commercially available from 3M) for delivery.Subcutaneous injection into the lesion (in normal saline or PBS) canalso be used

It is contemplated that any molecular biology, cellular biology orbiochemical technique known in the art may be used to generate and/ortest treatments contemplated herein. In addition, protein chemistrytechniques are contemplated to assess utility of treatments in modelsystems developed herein (e.g. mouse model system).

E. KITS

In certain embodiments, a kit contemplated herein may includecompositions discussed above for treating a subject having a conditioncontemplated herein, such as oral mucositis or psoriasis. The kits caninclude one or more containers containing the therapeutic compositions.Any of the kits will generally include at least one vial, test tube,flask, bottle, syringe or other container, into which compositions maybe preferably and/or suitably aliquoted. Kits herein may also include akit for assessing biological targets that contribute to a conditioncontemplated herein.

F. EXAMPLES

The following examples are included to illustrate various embodiments.It should be appreciated by those of skill in the art that thetechniques disclosed in the examples that follow represent techniquesdiscovered to function well in the practice of the claimed methods,compositions and apparatus. However, those of skill in the art should,in light of the present disclosure, appreciate that many changes may bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Smad7 transgenic mice have been described with multiple functions ofSmad7. This mouse model is crucial for understanding that Smad7 has apotent effect in oral mucositis. Smad7 promotes healing of oral wounds.Based on these findings, if Smad7 can provide protection from oralmucositis can be assessed. Smad7 transgenic mice and their littermateswere exposed to radiation. In non-transgenic littermates, all died oforal mucositis in 10 days due to loss of oral intake-associateddehydration and starvation. In contrast, none of the Smad7 transgenicmice formed oral mucositis (FIG. 1). Based on this data, localpharmacologic delivery of Smad7 can be used to treat oral mucositis. Inother embodiments, a Smad7 expression plasmid with eitherreplication-free viral vector or non-viral vector, can be used to treatoral mucositis.

Preliminary data show that Smad7 promotes healing mainly throughpromoting epithelial cell migration into the wounds and protecting cellsagainst DNA damage (FIG. 1), the latter is tumor suppressive. Smad7 hasbeen shown to inhibit cancer metastasis and unpublished data show Smad7overexpression in mice does not increase susceptibility to cancer. Giventhat unresolved inflammation is one of the major causes of failedhealing in oral mucositis, the strong effect of Smad7 can significantlypromote healing of oral mucositis. In one exemplary example, transgenicmice were generated with severe skin psoriasis, and these mice diewithin 6 months due to severe itch-associated wasting syndrome. Whenthese psoriasis mice were bred with Smad7 transgenic mice (the sameshowing resistance to radiation-induced oral mucositis in FIG. 1), skininflammation was absent and the mice lived a normal lifespan (FIG. 2).Interestingly, in both cases (FIGS. 1-2), Smad7 transgene expressionlevel is only 2-fold of endogenous Smad7, suggesting that a low dose ofSmad7 is sufficient for therapeutic effect. Smad7 oral patch/gel can beadministered by patients with minimal systemic effects.

The inventors generated a recombinant human Smad7 (98% homology to mouseSmad7) with an N-terminal Tat-tag (Tat-Smad7) that allows proteins topermeate the cell membrane and reach the nucleus in a matter of seconds(Cardarelli et al., 2008; Kalvala et al., 2010; Brooks et al., 2005).The human Smad7 cDNA nucleotide sequence was altered to optimize codonsfor bacterial protein production and a 5′ Tat-tag (9 aa) was added. TheTat.Smad7 was cloned into the pET101 protein expression vector(Invitrogen) for protein production in E. coli. The vector contains a V5epitope for protein identification using a V5 antibody and 6×His tag forprotein purification at the 3′ of the Tat-Smad7 protein. The inventorstested purified Tat-Smad7 protein transduction at a concentration of 7.5μg/ml. In less than 5 minutes after exposure to Tat-Smad7, nearly 100%of cells showed nuclear Tat-Smad7 (FIG. 3), which was retained at leastthe first 2 h after transduction. Later, Tat-Smad7 was also detected inthe cytoplasm (FIG. 3), consistent with its ability to move between thenucleus and the cytoplasm (Zhang et al., 2007). In reference to previousreports that cytoplasmic Smad7 blocks TGFβ receptor-mediated Smadphosphorylation and/or increases Smad degradation (Massague et al.,2005), Tat-Smad7 transduced keratinocytes abrogated Smad2phosphorylation (FIG. 3), suggesting it is functionally intact.Consistent with its long half-life, Tat-Smad7 was still detectable incells 36 hr after withdrawal of the 5-min Tat-Smad7 treatment (notshown). To determine if this is rapid enough for Tat-Smad7 to penetrateoral mucosa in vivo, the inventors treated 5 mice orally with 10 μg ofTat-Smad7 in 10 μl phosphate buffered saline (PBS) buffer once, with anhour food/water restriction after treatment, and excised oral mucosa forTat-Smad7 detection 24 h after treatment. Tat-Smad7 was detecteduniformly in epithelial and stromal cells of oral mucosa (FIG. 4). Thesedata suggest that local Tat-Smad7 delivery quickly achieves cellularuptake and should not require more than daily application.

Although the inventors have an endotoxin removal step in thepurification process, they further assessed whether Tat-Smad7 isfunctional in vivo, and if potential endotoxin contamination fromproduction in bacteria poses a toxicity risk. The inventorss.c.-injected Tat-Smad7 (10 μg/mouse, 3 times/wk) into K5.TGFβ1 micewhich exhibit severe skin inflammation (Li et al., 2004). In all three,K5.TGFβ1 mice in this pilot experiment, Tat-Smad7 treatmentsignificantly alleviated skin inflammation (FIG. 5) and no obvious sideeffects were observed. Immunofluorescence staining using a V5 antibodyshows that s.c.-injected Tat-Smad7 protein accumulated in the dermis andthroughout the epidermis (FIG. 5). Tat-Smad7-transduced K5.TGFβ1 skinshowed reduced pSmad2 and NFκB p50 in both the epidermis and the stroma(FIG. 5).

To test if Tat-Smad7 can treat radiation-induced oral mucositis throughoral delivery, the inventors irradiated mouse cranial facial area andtreated mice with Tat-Smad7 afterwards. Seven-to-nine week old C3Hfemale mice were anesthetized and exposed to 16Gy, 20Gy or 25Gy headirradiation with an RS2000 X-ray irradiator. Five days afterirradiation, mice were treated daily with either 10 μg Tat.Smad7dissolved in 50% glycerol/PBS or 50% glycerol/PBS (control), each groupcontained 4 mice, beginning on day 5 after irradiation when tissuedamage in oral mucosa occurs. Grossly, Tat-Smad7 treated mice recoveredbody weight more rapidly than control mice with 16Gy or 20Gy irradiation(FIG. 6); control mice with 25Gy irradiation were all euthanized forhumane reasons hence body weight recovery could not be monitored.Histology shows that by day 9 after 20Gy or 25Gy radiation, control micedeveloped oral mucositis (open ulcers, FIG. 7). In contrast, Tat-Smad7treated mice had damaged oral mucosa with thinning epithelial layers butno ulcer formation (FIG. 7). Continual treatment of 20Gy irradiated micewith Tat-Smad7 till day 13 accelerated repair. By day 14, when oralmucositis ulcers just finished re-epithelialization in control mice,oral epithelia in Tat-Smad7 treated mice was almost completely recoveredto normal morphology with only a few damaged epithelial cells and someinfiltrated leukocytes (FIG. 7). With 16 Gy irradiation, tissue damagewas most severe on day 9 in control mice with thinning and disorganizedepithelial layers (FIG. 7), however, this dose of radiation isinsufficient to cause oral mucositis and mice orally treated withTat-Smad7 from day 5 to day 8 retained essentially normal epithelium(FIG. 7).

Immunostaining using the antibody against the V5 tag of the Tat-Smad7shows that Tat-Smad7 protein was delivered primarily to oral epithelialcells (FIG. 8). Cells positive for pH2AX, a DNA damage marker, weresignificantly reduced in Tat-Smad7-treated oral mucosa (FIG. 8).Consistent with observed DNA damage, apoptotic cells, as determined byTUNEL assay, were also significantly reduced in Tat-Smad7 treated oralmucosa (FIG. 8). Infiltrated leukocytes, identified by CD45 staining,were prominent in irradiated oral mucosa treated with vehicle controlbut were significantly reduced in Tat-Smad7 treated mucosa (FIG. 8).CD31 staining to highlight vessels, shows enlarged vessels in irradiatedoral mucosa treated with vehicle control but normal vessel sizes inTat-Smad7 treated oral mucosa (FIG. 8).

In sum, these data show that Tat-Smad7 local delivery can treat severeoral mucositis through preventing tissue damage, improving epithelialhealing and reducing inflammation and vessel damage in the stroma.

The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter.

G. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method for treating or preventing an inflammatory condition in asubject comprising providing to the subject a therapeutically effectiveamount of a Smad7 (mothers against decapentaplegic homolog 7).
 2. Themethod of claim 1, wherein Smad7 is provided as a protein, an expressionvector encoding a Smad7 protein.
 3. The method of claim 2, wherein thecomposition comprises a Smad7 protein fused to a protein transductiondomain (PTD).
 4. The method of claim 3, wherein said Smad7 proteinfusion further comprises a regulatory domain that prevents Smad7function in the absence of an activator, and the method furthercomprises provision of said activator. 5-6. (canceled)
 7. The method ofclaim 1, wherein the Smad7 is provided in a formulation on a patch, in agelatinous composition, in a microsphere, in a microbead or combinationthereof.
 8. The method of claim 7, wherein the patch comprises abiodegradable patch.
 9. The method of claim 8, wherein saidbiodegradable patch comprises an alginate polymer.
 10. The method ofclaim 1, wherein the Smad7 is provided local to an affected region. 11.The method of claim 1, wherein the Smad 7 is provided systemically. 12.The method of claim 1, further comprising administering a secondanti-inflammatory therapy to said subject.
 13. The method of claim 12,wherein said PTD is Tat.
 14. The method of claim 1, wherein thecomposition comprises a detectable marker.
 15. The method of claim 1,wherein the acute inflammatory condition is selected from gut mucositis,oral mucositis, psoriasis, an autoimmune disease, acute wound, chronicwound, ulcer, trauma, surgery, chemotherapy, radiotherapy or cytokinetherapy. 16-25. (canceled)
 26. The method of claim 1, wherein theinflammatory condition is oral mucositis.
 27. The method of claim 26,further comprising administering a second mucositis therapy to saidsubject.
 28. The method of claim 25, wherein said second mucositistherapy is viscous 2% lidocaine, baking soda solution, saline solution,BAX solution (lidocaine, diphenhyramine, sorbitol and Mylanta), betacarotene, tocopherol, laser irradiation, silver-nitrate, misoprostol,leucovorin, systemic keratinocyte growth factor, pentoxifylline,allopurinol, systemic sucralfate, chlorhexidine gluconate orcryotherapy.
 29. The method of claim 26, wherein the subject comprises asubject having a transplant, a subject having cancer or a subject havinga condition requiring radiation therapy.
 30. The method of claim 29,wherein the subject has cancer and the cancer is selected from the groupconsisting of oral cancer, colon cancer, breast cancer, head and neckcancer, pancreatic cancer and other cancers treated with upper bodyradiation or repeated cycles of chemotherapy.
 31. The method of claim26, wherein the subject has undergone upper-body radiation.
 32. Themethod of claim 1, further comprising administering said composition tosaid subject a second time. 33-42. (canceled)
 43. A Smad7 protein fusedto a protein transduction domain (PTD).
 44. The Smad7 protein of claim43, wherein said PTD is Tat.
 45. The Smad7 protein of claim 43, whereinsaid protein is formulated with one or more pharmaceutically acceptableexcipients.
 46. The Smad7 protein of claim 43, wherein said one or morepharmaceutically acceptable excipients is selected for topicaladministration.
 47. The Smad 7 protein of claim 43, wherein said one ormore pharmaceutically acceptable excipients is selected for systemicadministration.